The present invention concerns a novel cytoskeletal protein, myotilin, which contains Ig-like domains homologous to a giant sarcomeric structural protein titin. Myotilin is expressed in skeletal and cardiac muscles, it colocalizes with xcex1-actinin in the sarcomeric I-bands and directly interacts with xcex1-actinin. Expression of myotilin in mammalian non-muscle cells and in yeast causes reorganization of actin into thick F-actin bundles and inhibits growth of yeast cells.
Among the various cell types in the higher organisms, the striated muscle cells have differentiated to carry out the task of force generation and transduction. To serve this very specialized function, the muscle cells express many gene products or mRNA splice variants that are not found in other cells of the body. Many of the muscle specific genes encode cytoskeletal proteins by which a highly organized sarcomeric architecture is created [1, 2]. The major components of thin and thick filaments, actin and myosin, are linked to a variety of molecules regulating the assembly, structural integrity and function of the striated muscle. For instance, the giant protein titin that spans from the M-line of the thick filament to the Z-line of the thin filament, functions as a spring and a ruler of the sarcomere, and xcex1-actinin, an actin-binding protein, crosslinks thin filaments into antiparallel bundles in the Z-lines [2-8]. The force generated by cytoskeletal components of the contracting subunits is transduced through the plasma (sarcolemma) membrane to the extracellular matrix via a connecting multi-subunit dystrophin-glycoprotein complex [9, 10].
The importance of the individual components of the sarcomeric and sarcolemmal structures is highlighted by recent findings demonstrating that mutations in several different structural proteins result in muscular diseases such as muscular dystrophies and cardiomyopathies [9-12]. Many of the identified muscle disease genes encode proteins of dystrophin-associated sarcolemmal complex, but recently also other types of molecules, including regulators of the sarcomeric architecture, have been indicated to participate in pathogenesis of certain disease forms. A mutation in xcex1-tropomyosin gene, TPM3, was shown to cause an autosomal dominant nemaline myopathy (NEM1) [13]. The nebulin gene is a candidate for another form of nemaline myopathy (NEM2) [14] and the titin gene is a candidate for autosomal dominant tibial muscular dystrophy [15].
In spite of recent advances, several clinically distinguishable forms of muscular dystrophy with unidentified disease genes exist. Two forms of muscular dystrophy, a dominant form of limb-girdle muscular dystrophy (LGMD1A) and a dominant form of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) have been mapped to an overlapping locus in 5q31 [16,17].
Several studies have shown that actin cytoskeleton is substantially modified in transformed cells [reviewed in 18, 19 and 20]. In cells, actin molecules undergo dynamic reorganization, i.e. polymer formation from actin monomers and disruption or modulation of existing polymers. These events are controlled by a variety of actin-binding proteins with versatile activities. The complex dynamic regulation of cytoskeletal filaments depends on the expression and activity of various components within cells. Interestingly, a large fraction of actin exist in most cell types as monomers, whereas in muscle cells more than 99% of actin is in filaments. This suggests that muscle cells express protein(s), some of which may be unknown, whose function is to preserve the actin molecule equilibrium in a polymerized state. Taken the important role of actin cytoskeleton in functions related to abnormal cell growth and the changes in actin organization in transformed cells, factors regulating actin organization serve as attractive targets for cancer chemotherapy. Such an idea has been recently supported by experimental data indicating that two novel actin-stabilizing components, jasplakinolide and chondramides inhibit growth of transformed cells [20, 21].
Here we describe the cDNA sequence and structure of myotilin gene, which encodes a novel component of the striated and cardiac muscle cytoskeleton. Myotilin protein contains two C2-type Ig-like domains with considerable homology to certain Ig-domains of titin. Myotilin resides both in the sarcomere, where it localizes within the I-bands and is bound to xcex1-actinin, and along the sarcolemmal membrane. The myotilin gene locates in chromosome 5q31 inside a 2Mb region, which contains the LGMD1A disease gene [16], and thus is a candidate for LGMD1A. Transfection of myotilin into mammalian cells and yeast cells induces formation of thick actin bundles and reduces growth of yeast, indicating a role for myotilin in organization of the actin-containing cytoskeleton.
The designation xe2x80x9cmyotilinxe2x80x9d comes from myofibrillar protein with titin-like Ig-domains. It should be noted that the designation xe2x80x9cmyofilinxe2x80x9d which was used earlier, was amended due to the fact that a muscle-specific gene of Echinococcus granulosus was previously termed xe2x80x9cmyophilinxe2x80x9d [22]. Although the terms are spelled differently, a similar pronunciation creates possibility for confusion and therefore the designation xe2x80x9cmyofilinxe2x80x9d was changed to xe2x80x9cmyotilinxe2x80x9d.
Expression of myotilin in mammalian cells and yeast causes reorganization of actin into thick filaments. The expression of myotilin or its C-terminal fragment (amino acids 215-498) changes yeast morphology and reduces growth rate. The actin-organizing and growth inhibiting properties suggest that myotilin or its fragments may be used in development of substances to control cell growth in various pathological conditions including treatment of cancer or microbial infections.